Novel mutants of acetyl-transferase sb-atf

A process for production of retinol acetylating enzymes, preferably acetyl transferase of class [EC 2.3.1.84], having increased activity towards acetylation of retinol into retinyl acetate, said process comprising:

• (a) providing a fungal enzyme involved in acetylation of retinol, particularly ATF originated from Saccharomyces, wherein said non-modified enzyme expressed in a suitable host cell and under suitable culture conditions is capable of producing up to 10 wt % retinyl acetate from retinol based on total retinoids produced by said host cell;
• (b) introduction of at least 2 amino acid substitutions on positions corresponding to L34F and M434V in the polypeptide according to SEQ ID NO:1, furthermore introduction of at least 1 to 7 amino acid substitution(s) into the enzyme of (a) to generate a modified enzyme involved in acetylation of retinol, wherein the ability to convert retinol into retinyl acetate is increased by at least about 10%, such as e.g. by at least 10 to 760% when compared to the acetylation of the respective non-modified enzyme of step (a);
• wherein the at least 1 or 7 amino acid substitution(s) are being located at position(s) corresponding to amino acid residue(s) being selected from the group consisting of position 35, 346, 371, 373, 419, 437, 478 in SEQ ID NO:1 and wherein the substitute amino acid(s) is/are different from Y35, F346, V371, F373, N419, R437, W478 and/or combinations thereof, wherein the amino acid residue to be introduced at position corresponding to position 35, or 478 in SEQ ID NO:1 is preferably phenylalanine, and/or wherein the amino acid residue to be introduced at position corresponding to position 346 or419 in SEQ ID NO:
• 1 is preferably leucine, and/or wherein the amino acid residue to be introduced at position corresponding to position 371 in SEQ ID NO:1 is preferably isoleucine, and/or wherein the amino acid residue to be introduced at position corresponding to position 373 in SEQ ID NO:
• 1 is preferably alanine, and/or wherein the amino acid residue to be introduced at position corresponding to position 437 in SEQ ID NO:1 is preferably valine.

A modified enzyme involved in acetylation of retinol into retinyl acetate,

• particularly fungal enzyme comprising at least 2 modifications, such as amino acid substitutions L34F and M434V being located at positions corresponding to amino acid residues 34 and 434 in the polypeptide according to SEQ ID NO:1, further comprising at least 1, 2, 3, 4, 5, 6, 7 amino acid substitution(s), said amino acid substitution(s) being located at position(s) corresponding to amino acid residue(s) being selected from the group consisting of position 35, 346, 371, 373, 419, 437, 478 and combinations thereof, in the polypeptide according to SEQ ID NO:1 or a polypeptide with a sequence of at least about 20%, such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:1, and wherein the substitute amino acid residue(s) are different from amino acids corresponding to Y35, F346, V371, F373, N419, R437, W478 and combinations thereof in the enzyme according to SEQ ID NO:
• 1, preferably wherein the amino acid residue(s) to be substituted are corresponding to residues selected from the group consisting of Y35, F346, V371, F373, N419, R437, W478 and combinations thereof in a polypeptide according to SEQ ID NO:1, particularly wherein the substitute amino acid residue to be introduced at position corresponding to position 35, or 478 in SEQ ID NO:1 is preferably phenylalanine, wherein the substitute amino acid residue to be introduced at position corresponding to position 346 or419 in SEQ ID NO:
• 1 is preferably leucine, wherein the substitute amino acid residue to be introduced at position corresponding to position 371 in SEQ ID NO:1 is preferably isoleucine, wherein the substitute amino acid residue to be introduced at position corresponding to position 373 in SEQ ID NO:
• 1 is preferably alanine, and/or wherein the substitute amino acid residue to be introduced at position corresponding to position 437 in SEQ ID NO:1 is preferably valine.